The present invention relates to a process for the production of cyclodextrins.
In particular, the invention relates to a process for the production of cyclodextrins by reaction of starch with cyclodextrin-glycosyl-transferase (CGTase) which is produced by Bacillus ohbensis, deposited with the Fermentation Research Institute in Japan (FRI), 1-3 Higashi-1-Chome, Tsukuba-Shi, Japan, under the Budapest Treaty, previously designated under deposit number FERM P-1990, and since 1981 designated FERM BP-3180, or produced by a mutant derived from Bacillus ohbensis (e.g., FERM BP-3180), or produced by any other strain in which the CGTase gene from B. ohbensis has been cloned. This enzyme reacts with and degrades starch to form cyclodextrins. The thusformed cyclodextrins (designated hereinbelow by CD), e.g., .beta.-cyclodextrin (.beta.-CD) and .gamma.-cyclodextrin (.gamma.-CD) are then isolated.
.beta.-CD is a cyclic molecule consisting of 7 glucopyranose units linked to each other by 1.fwdarw.4 bonds; .gamma.-CD is a cyclic molecule consisting of 8 glucopyranose units linked to each other by 1.fwdarw.4 bonds. These molecules possess a central hydrophobic cavity which enables the production of inclusion compounds with host molecules. The binding of the inclusion compounds occurs with low energy; thus, the inclusion phenomenon is reversible. The CDs have a particularly interesting field of application, e.g., for the encapsulation of aromas, vitamins, colors, fatty acids, pharmaceutical active principles, pesticides and fungicides. The production of .beta.-CD and .gamma.-CD using the CGTase produced is known.
A new .alpha.-amylase, i.e., the CGTase produced by Bacillus ohbensis, was first described in Japanese Patent 902415 (Japanese Application No. 7335774). Moreover, this patent discloses the preparation of .beta.-CD and .alpha.-CD (a cyclic molecule consisting of 6 glucopyranose units linked to each other by 1.fwdarw.4 bonds) by reaction of liquefied or hydrolyzed starch with the CGTase produced by Bacillus ohbensis.
The article entitled "Determination of CGTase from Bacillus ohbensis and its optimum pH using HPLC", Sato, M., et al., Agricultural and Biological Chemistry, 49 (4), 1189-1991 (1985) discloses that the optimum pH of the CGTase produced by Bacillus ohbensis is pH 5.5. This article also specifies that the analysis by high pressure liquid chromatography (HPLC) permits a direct measurement of the cyclodextrin formed and that this method of analysis is also the most reliable.
The article entitled "Comparative studies of CGTases from Bacillus ohbensis, Bacillus macetans and Bacillus circulans and production of cyclodextrins using those CGTases", Yagi, Y., et al., J. Jpn. Soc. Starch Sci., 33, 144-151 (1986), discloses the following information:
the optimum pH for the CGTase produced by PA1 Bacillus ohbensis is pH 5.5; PA1 the CGTase produced by Bacillus ohbensis put into reaction with liquid starch produces .beta.-CD and .gamma.-CD; PA1 with liquefied starch and CGTase produced by Bacillus ohbensis, a yield of 21.6% by weight of CD is obtained at pH values between 5 and 10. PA1 the temperature, pH and concentration of starch, agitated in water with the B.O. enzyme is adjusted to the desired values, thus ensuring optimum control of the process parameters and avoiding, due to regulation of starch input, the formation of a gel or aggregate in the reactor; PA1 when the desired viscosity is reached, the ultrafiltration device is set in operation and the concentration of the reactants in the reactor adjusted by adding water, and the thus-produced cyclodextrins are continuously extracted in the permeate; it is not necessary to add more starch, resulting in a gradual decrease in the reaction volume in the reactor.
EP-A-220719 describes a process for the production of cyclodextrins (.alpha., .beta.and .gamma.) according to which liquefied starch is subjected to CGTase produced by Bacillus ohbensis with a pH preferably maintained between 6.5 and 7.5.
The cyclodextrins formed by this reaction are typically extracted from the reaction mixture by adsorption on a water insoluble resin, the ligand of which has such a size to be included in the cyclodextrins, and in that way, forms inclusion compounds.
This process favors the formation of cyclodextrins as well as the purification of these pro-ducts which are then eluted with warm water or with a mixture of water and alcohol: however, this extraction process is expensive and complicated, and the concentration of the cyclodextrins of these aqueous solutions is very low; it does not exceed 8 g/l.